Multichannel stimulation module as a tool for animal studies on cortical neural prostheses.

Intracortical microstimulation to the visual cortex is thought to be a feasible technique for inducing localized phosphenes in patients with acquired blindness, and thereby for visual prosthesis. In order to design effective stimuli for the prosthesis, it is important to elucidate relationships between the spatio-temporal patterns of stimuli and the resulting neural responses and phosphenes through pre-clinical animal studies. However, the physiological basis of effective spatial patterns of the stimuli for the prosthesis has been little investigated in the literature, at least partly because that the previously developed multi-channel stimulation systems were designed specifically for the clinical use. In the present, a 64-channel stimulation module was developed as a scalable tool for animal experiments. The operations of the module were verified by not only dry-bench tests but also physiological animal experiments in vivo. The results demonstrated its usefulness for examining the stimulus-response relationships in a quantitative manner, and for inducing the multi-site neural excitations with a multi-electrode array. In addition, this stimulation module could be used to generate spatially patterned stimuli with up to 4,096 channels in a dynamic way, in which the stimulus patterns can be updated at a certain frame rate in accordance with the incoming visual scene. The present study demonstrated that our stimulation module is applicable to the physiological and other future studies in animals on the cortical prostheses.

Role of basal sweating in maintaining skin hydration in the finger: A long-standing paradox in dry skin resolved.

A long-standing paradox in dermatology is why skin dehydration in the fingers can be triggered by repeated water exposure despite the action of water to hydrate skin tissue. Potential clues might be provided by identifying a mechanism through which water is held in the skin of the fingers. We speculated that this mechanism would be impaired after repeated water exposure. Here, we investigated whether there might be glabrous skin-specific water-holding machinery and whether this machinery might be impaired in dry skin/hand eczema. We examined this by using an impression-mould technique, allowing for an accurate quantification of sweat gland/duct activity and optical coherence tomography. Unlike in hairy skin, sweat pores were rarely detected at the folds of the finger at baseline. Surprisingly, after water exposure, sweat pores at the folds opened and those at the ridges closed in healthy controls (HCs). Sweating in the dermal folds of the hands correlated with skin hydration, and decreased in dry skin/hand eczema, suggesting that its impairment may be one of the causes of dry skin. After repeated water exposure, basal sweating response at the folds was exhausted in patients with dry skin/hand eczema as well as HCs. This exhaustion was rescued by exposing individuals to high humidity. Basal sweating defects would be a target for dry skin/hand eczema. Maintaining basal sweating responses in the finger is the best preventive measures in achieving prevention of dry skin/hand eczema.

Identification of chemical compounds as an inhibitor of mitochondrial ATP synthesis, leading to an increased stress resistance and an extended lifespan in C. elegans.

It is well known that the disruption of the mitochondrial respiratory components prolongs lifespan in many species. The mitochondrial stress response can lead to an increased survival rate through the restoration of the cellular homeostasis. Therefore, developing pharmacological interventions that induce mitochondrial stress response may be desirable to delay the onset of age-related diseases and promote a healthy life. In this study, we present chemical compounds, revealed by systematic screening of chemical libraries, which inhibit mitochondrial ATP synthesis in mammalian cells. Our study demonstrates that these compounds alter the body length and promote the oxidative stress response which leads to an increased longevity in Caenorhabditis elegans. Thus, our study identifies chemical compounds that may have potential therapeutic applications through affecting the mitochondrial function.

Prurigo nodularis as a sweat gland/duct-related disorder: resolution associated with restoration of sweating disturbance.

Little attention has been given to the involvement of sweat glands/ducts in the pathogenesis of prurigo nodularis (PN). According to recent studies, PN is likely to develop under conditions characterized by dry skin, such as atopic dermatitis (AD), suggesting a strong impact of skin dryness on PN development. No therapeutic modalities produced complete resolution of PN without exacerbations. We previously reported that increases in skin dryness by sweating disturbance could initiate the development of AD. We investigated whether sweating responses were impaired in refractory PN lesions; and, if so, we asked whether the PN lesions could resolve by restoring sweating disturbance. Using the impression mold technique, which allows an accurate quantification of individual sweat gland/duct activity, we examined basal sweating under quiescent conditions and inducible sweating responses to thermal stimulus in PN lesions and normal-appearing skin in the same patients before and after treatment with a moisturizer or topical corticosteroids. Sweating disturbance, either basal or inducible, was most profoundly detected in the "hub" structure corresponding to the center of PN papule before the treatment. This sweating disturbance was immunohistochemically associated with the leakage of sweat into the dermis. This disturbance was restored by treatment with a moisturizer. Our limitations include a relatively small patient cohort and lack of blinding. Sweating disturbance could be one of the aggravating factors of PN development. Refractory PN with low skin hydration may resolve by restoring sweating disturbance.

Focal activation of neuronal circuits induced by microstimulation in the visual cortex.

OBJECTIVE: Microstimulation to the cortical tissue applied with penetrating electrodes delivers current that spreads concentrically around the electrode tip and is known to evoke focal visual sensations, i.e. phosphenes. However, to date, there is no direct evidence depicting the spatiotemporal properties of neuronal activity induced immediately after microstimulation and how such activity drives the subsequent local cortical circuits. APPROACH: In the present study, we imaged the spatiotemporal distribution of action potentials (APs) directly induced by microstimulation and the subsequent trans-synaptic signal propagation using a voltage-sensitive dye (VSD) and a calcium-sensitive dye (CaSD) in slice preparations of the mouse primary visual cortex. MAIN RESULTS: The directly induced APs were confined to the close vicinity of the electrode tip, and the effective distance of excitation was proportional to the square root of the current intensity. The excitation around the electrode tip in layer IV mainly propagated to layer II/III to further induce the subsequent focal activation in downstream local cortical circuits. The extent of activation in the downstream circuits was restrained by competitive interactions between excitatory and inhibitory signals. Namely, the spread of the excitation to lateral neighbor neurons along the layer II/III was confined by the delayed inhibition that also spread laterally at a faster rate. SIGNIFICANCE: These observations indicate that dynamic interactions between excitatory and inhibitory signals play a critical role in the focal activation of a cortical circuit in response to intracortical microstimulation and, therefore, in evoking a localized phosphene.

A single retinal circuit model for multiple computations.

Vision is dependent on extracting intricate features of the visual information from the outside world, and complex visual computations begin to take place as soon as at the retinal level. In multiple studies on salamander retinas, the responses of a subtype of retinal ganglion cells, i.e., fast/biphasic-OFF ganglion cells, have been shown to be able to realize multiple functions, such as the segregation of a moving object from its background, motion anticipation, and rapid encoding of the spatial features of a new visual scene. For each of these visual functions, modeling approaches using extended linear-nonlinear cascade models suggest specific preceding retinal circuitries merging onto fast/biphasic-OFF ganglion cells. However, whether multiple visual functions can be accommodated together in a certain retinal circuitry and how specific mechanisms for each visual function interact with each other have not been investigated. Here, we propose a physiologically consistent, detailed computational model of the retinal circuit based on the spatiotemporal dynamics and connections of each class of retinal neurons to implement object motion sensitivity, motion anticipation, and rapid coding in the same circuit. Simulations suggest that multiple computations can be accommodated together, thereby implying that the fast/biphasic-OFF ganglion cell has potential to output a train of spikes carrying multiple pieces of information on distinct features of the visual stimuli.

Imaging of population spikes induced by repetitive stimulus pulses in mouse cerebral slices in vitro.

Effects of the repetitive current pulses of microstimulation on spatio-temporal neuronal excitations in the primary visual cortex in mouse cerebral slices in vitro were examined by utilizing the voltage-sensitive dye imaging technique. The amplitude and spatial extent of the population spike directly induced by the stimulus pulse was significantly reduced in response to successive stimulus pulses at 200 Hz. This suggested that the high-frequency microstimulation may not be efficient for inducing the neuronal spiking, at least, in vitro.

Age-related changes in the spatiotemporal responses to electrical stimulation in the visual cortex of rats with progressive vision loss.

The Royal College of Surgeons (RCS) rat gradually loses vision due to retinal degeneration. Previous physiological studies have depicted the progressive loss of optical responses in the visual pathway, including the primary visual cortex (V1), over the course of retinal degeneration in the RCS rat. However, little is known about how the excitability of the V1 circuit changes during over the course of the gradual loss of visual signal input from the retina. We elucidated the properties of responses to electrical stimulations directly applied to V1 at different stages of vision input loss in the RCS rat in reference to those of the Long-Evans (LE) rat, using in vivo voltage-sensitive dye imaging. The V1 neuronal network of the RCS rat exhibited an excitatory response comparable to the LE rat. The excitatory response was maintained even long after total loss of the visual signal input from the retina. However, the response time-course suggested that the suppressive response was somewhat debilitated in the RCS rat. This is the first experiment demonstrating the long-term effect of retinal degeneration on cortical activities. Our findings provide the physiological fundamentals to enhance the preclinical research of cortical prostheses with the use of the RCS rat.

Retinal Circuit Emulator With Spatiotemporal Spike Outputs at Millisecond Resolution in Response to Visual Events.

To gain insights on how visual information of the real world is filtered, compressed, and encoded by the vertebrate retinas, emulating in silico the spatiotemporal patterns of the graded and action potentials of neuronal responses to natural visual scenes on biological time scale is a feasible approach. As a basic platform for such an emulation, we here developed a compact hardware system comprising an analog silicon retina and a field-programmable gate array module. With utilizing the Izhikevich formalism, a retinal circuit model that emulates spiking of ganglion cells was implemented in this system. The emulated spike timing had the resolution of about 2 ms relative to the stimulus onset and was little affected by timings of the synchronous frame sampling in the silicon retina. Thus, the emulator can mimic the event-driven spike outputs of biological retinas. The system was useful for simultaneously visualizing neural images of both the graded potentials and the spikes in response to real live visual scenes. Since our emulator system is reconfigurable, it provides a flexible platform for investigating visual functions of retinal circuits under natural visual environment.

A large scale simulation of excitation propagation in layer 2/3 of primary and secondary visual cortices of mice.

Analyzing network architecture and spatio-temporal dynamics of the visual cortical areas can facilitate understanding visual information processing in the brain. Recently, several physiological experiments utilizing the fast in-vivo imaging technique have demonstrated that the primary visual cortex (V1) and the secondary visual cortex (V2) in mice exhibit complex properties of the responses to visual and electrical stimuli. In order to provide a tool for quantitatively analyzing such a complex dynamics of the cortices at the level of neurons and circuits, here, we constructed a physiologically plausible large-scale network model of the layers 2/3 of V1 and V2, composed of 14,056 multi-compartment neuron models. The Message-Passing-Interface-based parallel simulations of our network model were able to reproduce, at least quantitatively, the neural responses experimentally observed in mouse V1 and V2 with the voltage-sensitive dye imaging.

Cortical neural excitations in rats in vivo with using a prototype of a wireless multi-channel microstimulation system.

Understanding neural responses to multi-site electrical stimuli would be of essential importance for developing cortical neural prostheses. In order to provide a tool for such studies in experimental animals, we recently constructed a prototype of a wireless multi-channel microstimulation system, consisting of a stimulator chip, wireless data/power transmitters and receivers, and microcomputers. The proper operations of the system in cortical neural excitations were examined in anesthetized rats in vivo, with utilizing the voltage-sensitive dye imaging technique.

Voltage-sensitive dye imaging of the visual cortices responding to electrical pulses at different intervals in mice in vivo.

Properties of the neural responses to electrical stimulus pulses delivered at various inter-pulse intervals were examined in the visual cortices of mice in vivo, with utilizing the voltage-sensitive dye imaging technique. Our experimental results provided the relationships between the inter-pulse intervals and the stimulus-evoked transient depolarizations, which may offer insight into the design of effective and efficient stimulation for cortical visual prostheses.

A multichannel current stimulator chip for spatiotemporal pattern stimulation of neural tissues.

We developed a prototype very-large-scale integration chip of a multichannel current stimulator for stimulating neural tissues by utilizing 0.25 µm high-voltage complementary metal-oxide-semiconductor technology. Our designed chip has 20 output channels that are driven by five current buffers arranged in parallel; each buffer controls four output channels in time-sharing mode. The amplitude of a stimulation pulse can be controlled within a range of approximately ±100 µA/phase in each output channel. The stimulus parameters, e.g., amplitude and duration, are controlled separately for each channel by digital codes stored in built-in registers. Combinations of anode and cathode electrodes to pass the current can be changed online. We integrated our stimulator chip with a multielectrode array and studied the neuronal responses to multichannel current stimulations with various temporal patterns in mouse brain slices.

Interfacing neurons through the patch membrane pierced with single-walled carbon nanotubes.

The usability of single-walled carbon nanotubes (CNTs) as electrically conductive channels across the cell membrane was examined at the submicroscopic level. By using the patch-clamp technique, we found the surface-modified single-walled CNTs dispersed in the micropipette solution can provide an electrical access to the intracellular space across the patch of cell membrane in dissociated mammalian neurons, thereby enabling us to measure the membrane excitabilities in the 'pierced-patch' whole-cell mode.

A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T-lymphocytes reactive to tumor cells.

The present study attempted to identify a useful tumor-associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45-like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome-wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non-cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti-tumor effects of cytotoxic T-lymphocytes (CTL) specific to CDC45L-derived peptides induced from HLA-A24 (A*24:02)-positive donors. We identified three CDC45L-derived peptides that could reproducibly induce CDC45L-specific and HLA-A24-restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA-A24. In addition, we found that CDC45L (556) KFLDALISL(564) was eminent in that it induced not only HLA-A24 but also HLA-A2 (A*02:01)-restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L-specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L-derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy.

Peptides derived from human insulin-like growth factor-II mRNA binding protein 3 can induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer cells.

Insulin-like growth factor-II mRNA binding protein 3 (IMP-3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP-3 that can induce tumor-reactive and human leukocyte antigen (HLA)-A2 (A*02:01)-restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP-3-derived peptides predicted to bind to HLA-A2 were analyzed to determine their capacity to induce HLA-A2-restricted T cells in HLA-A2.1 (HHD) transgenic mice (Tgm). We found that three IMP-3 peptides primed HLA-A2-restricted CTL in the HLA-A2.1 Tgm. Among them, human CTL lines reactive to IMP-3 (515) NLSSAEVVV(523) were reproducibly established from HLA-A2-positive healthy donors and lung cancer patients. On the other hand, IMP-3 (199) RLLVPTQFV(207) reproducibly induced IMP-3-specific and HLA-A2-restricted CTL from healthy donors, but did not sensitize CTL in the HLA-A2.1 Tgm. Importantly, these two IMP-3 peptide-specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP-3 and HLA-A2. Cytotoxicity was significantly inhibited by anti-HLA class I and anti-HLA-A2 monoclonal antibodies, but not by the anti-HLA-class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP-3 protein was confirmed by specific killing of HLA-A2-positive IMP-3-transfectants but not the parental IMP-negative cell line by peptide-induced CTL. This suggests that these two IMP-3-derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy.

A new method for localizing the sources of correlated cross-frequency oscillations in human brains.

Anatomically distributed areas are dynamically linked to form functional networks for processing and integrating the different modalities of information in the human brain. A part of such networks is considered to be realized with synchronization of neuronal activities, which can generate correlated neural oscillation at the same and/or different frequency bands. To investigate the networks with the synchronization, analysis of connectivity between not only same frequency oscillation but also different frequency (i.e. cross-frequency) is needed. For source estimation with electroencephalogram (EEG) or magneto-encephalogram (MEG) signals, a spatial filtering technique is recently applied as an alternative method for equivalent current dipole (ECD) estimation technique. Non-adaptive type of spatial filtering technique, such as the Standardized low-resolution brain electromagnetic tomography (sLORETA), is reported to discriminate correlated sources. However, it may lead to inaccurate results due to its low spatial resolution. In the present study, we proposed a new systematic approach for localizing the sources of correlated cross-frequency oscillations. The method we propose can overcome the limitation of the non-adaptive spatial filtering technique by proactively using identified information in sensor level analysis (e.g. cross-correlation map and correlation topography), which allow us to focus on target sources. The performance of our proposed method is evaluated with simulated EEG signals, and is compared with traditional method.